Extended single-dose toxicity study of [²¹¹At]NaAt in mice for the first-in-human clinical trial of targeted alpha therapy for differentiated thyroid cancer

In recent years, radionuclide therapy is attracting attention for the treatment of various types of cancers. Among them, targeted alpha therapy with an alpha emitter is promising, with higher therapeutic effect than conventional beta radionuclide therapy [1]. Astatine (²¹¹At) is an alpha emitter that can be produced by the ²⁰⁹Bi(α, 2n)²¹¹At reaction using cyclotron [2]. Recently, we reported the excellent anti-tumor effect of [²¹¹At]sodium astatide ([²¹¹At]NaAt) in a preclinical study using a xenograft model of differentiated thyroid cancer [2]. We are now preparing for an investigator-initiated clinical trial of targeted alpha therapy for differentiated thyroid cancer refractory to standard ¹³¹I treatment or tyrosine kinase inhibitor, in consultation with the Pharmaceuticals and Medical Devices Agency (PMDA), the Japanese authority for drug approval. As there is no report on the intravenous administration of [²¹¹At]NaAt in humans, it is necessary to carefully perform a preclinical safety assessment for the first-in-human (FIH) clinical trial. Fortunately, alpha therapy using ²²³Ra (Xofigo) has already been approved in Japan, and its safety information can be used as a reference, to some extent [3]. However, as there is no description of radiopharmaceuticals in the official guidelines (ICH M3), which can be used as an official reference for PMDA, it was necessary to determine the optimum evaluation time point with reference to published reports with adequate evidence [4‐6].

In addition, there is no preclinical safety testing in association with GLP facilities capable of handling alpha-emitting nuclides in Japan. Thus, a toxicity study must be performed in our institution under the reliability standard based on Article 43 of the Japanese Pharmaceutical Law Enforcement Regulations with the support of contractors with adequate GLP compliance experience. As the permitted amount of ²¹¹At in preclinical radioactive controlled areas is limited by the Radiation Hazard Prevention Act, it is difficult to conduct toxicity studies in rats, dogs, and monkeys. In consultation with the PMDA, it was determined that the [²¹¹At]NaAt clinical trial can begin on the premise that it should be carried out with extreme caution so long as no obvious safety issues arise during the extended single-dose toxicity study in mice. Hence, the current study is an extended single-dose toxicity examination using mice with reference to the ICH guidelines and previous reports, which was performed in accordance with the reliability standard as a preclinical safety assessment of [²¹¹At]NaAt to determine the FIH dose [4‐7].

In the extended single-dose toxicity study of [²¹¹At]NaAt using mice conducted under a reliability standard, administration of [²¹¹At]NaAt at high doses (50 MBq/kg) resulted in weight loss, transient bone marrow suppression, and pathological changes in the testis. In the evaluation of body weight change, the 50 MBq/kg group showed a decreasing tendency from days 3 to 5 after administration, as confirmed in a previous study [5]. Blood examination revealed a transient decrease in WBC and platelet counts was observed in the 50 MBq/kg group (males and females) on day 5 compared to those of the control group, however, they recovered on day 14. This result was also consistent with that of a previous study [5]. In addition, TP, ALB, and TCHO were significantly higher in the male and female 50 MBq/kg groups on day 14 than in the control group, which was confirmed in previous studies and considered to reflect hypothyroidism via thyroid ablation [5]. Meanwhile, the decrease in TG (day 5 in 5 and 20 MBq/kg male, and 50 MBq/kg female), which was transiently observed followed by subsequent recovery on day 14, may have been related to the acute lipid metabolism disorder caused by hypothyroidism. In addition, the pathological significance of decreased LDH (day 5, 50 MBq/kg male), AST and TBIL (day 14, 50 MBq/kg male), AST and AMY (day 14, 50 MBq/kg female) and ALP (day 5, 50 MBq/kg female) is unknown as an abnormality or dysfunction is generally considered when these values are significantly elevated beyond the normal range.

In the organ weight measurement, the decrease in spleen weight on day 5, which was primarily observed in the 50 MBq/kg group, may be due to the effect of [²¹¹At] NaAt, as it is an organ with high physiological accumulation [5]. The weight of the salivary glands also tended to decrease in a dose-dependent manner, mainly at the concentration of 50 MBq/kg, however, no related abnormalities were detected in the histopathological examination. Moreover, considering that the physiological accumulation of [²¹¹At]NaAt and [¹³¹I]NaI treatment reportedly exerts adverse effects on the salivary gland, such as swelling, dysgeusia, and decreased salivary secretion, caution must be exercised when conducting clinical trials for adverse effects on the salivary glands. Meanwhile, the testes showed a significant weight decrease in the 50 MBq/kg group on day 14, and histopathological changes (appearance of multinucleated giant cells) were considered to be the effect of [²¹¹At]NaAt. However, no clear histological finding was observed in spermatogenesis compared to the control group. Since the formation of multinucleated giant cells was relatively mild, it is considered that these changes were not significant enough to induce histological changes in spermatogenesis. Although no significant abnormality was observed in the ovary, there was a significant weight decrease in the 50 MBq/kg group on day 14. As the reproductive organs are highly radiosensitive among the major organs, it is necessary to pay particular attention to fertility, and patients who desire to preserve fertility should be excluded from the clinical trial of [²¹¹At]NaAt. Furthermore, the decrease in heart weight on day 14 may represent a secondary effect of the hypometabolism or lipid metabolism disorder which caused the change in TCHO by thyroid ablation, as radiation-induced toxicity is uncommon in the heart. Alternatively, the observed decrease in kidney weight on day 14 must be monitored for potential radiation-induced toxicity; however, this may have also been a secondary effect as no significant corresponding histopathological changes were observed. In fact, the reference standard for evaluation of toxicity is histopathological examination. Thus, considering that these changes in weight were minimal, they might reflect other conditions or metabolic changes induced by thyroid ablation.

Histopathological examination showed colloidal depletion, degeneration/necrosis of follicular epithelial cells, and inflammatory cell infiltration in the thyroid gland on days 5 and 14, and there are well-known ablation effects. In addition, squamous epithelial cysts, gastric gland dilatation, and inflammatory cell infiltration were observed in the stomach on days 5 and 14, however, these changes were considered spontaneous lesions as they were not dose-dependent and were also observed in the control group. On days 5 and 14, vacuolation in the renal tubules of the outer renal medullary layer was observed in the kidneys of the female 50 MBq/kg group. Although vacuolation was observed only in the high-dose group, it has been reported in the renal tubules of the outer medullary layer in ICR mice [9]. In addition, as the findings on day 14 were similar to those on day 5 and no necrotic changes were observed on day 5, it was considered that the change was spontaneous. On day 5, the accessory adrenocortical tissue was observed under the adrenal capsule in the adrenal glands of the female 5 MBq/kg group, which was considered to be a spontaneous lesion as it is known as a tissue dysplasia during the embryonic period in mice [10]. In addition, on day 14, degeneration/necrosis, mineral deposits, multinucleated giant cells, yellowish-brown pigmentation, and fibrosis were observed in the adrenal cortex of one mouse in the male 50 MBq/kg group. As it was accompanied by chronic changes such as mineral deposition and fibrosis, and only one male case presented these symptoms on day 14, it was considered to be a spontaneous lesion. On day 5, sperm stasis and hypoplasia were observed in one mouse of the 5 and 50 MBq/kg groups, however, sperm stasis was observed only in the 20 MBq/kg group. Testicular hypoplasia was considered a spontaneous lesion as it was likely to have occurred before the administration of the test substance considering the degree of testicular hypoplasia was strong despite no necrotic changes. In addition, the testicular weight of the 50 MBq/kg group on day 5, which showed testicular hypoplasia, did not differ significantly from that of the control group, and is, therefore, considered to be spontaneous in combination with histological findings. On day 5, the epididymal tail was red in one mouse in the 5 MBq/kg group at necropsy, and degeneration/necrosis of the epididymal epithelium, fibrosis, and interstitial mononuclear cell infiltration were observed. This change was observed in only one mouse in the low-dose group and did not occur in the higher dose groups, suggesting that it was a spontaneous lesion unrelated to test substance administration. An intra-abdominal mass was found in one male and one female in the 5 MBq/kg group by necropsy on day 5. In mice, it is known that the entire lobe can be infarcted due to torsion of the lobe base of the caudate lobe [11], and the intra-abdominal mass observed in this study may have been formed in association with the infarction. As it occurred in only one case in each group and did not occur in the higher dose groups, it was considered a spontaneous lesion unrelated to administration.

Regarding the comparison of absorbed doses in each organ, which was calculated in our previous study, the highest absorbed dose was observed in the thyroid gland (5691 mGy/MBq), followed by the stomach (3941 mGy/MBq) and bladder (1955 mGy/MBq) [5]. Those pertaining to the thyroid agree with the current study results, as the ablation effect was observed in all groups (> 5 MBq/kg). In the stomach, accumulation of ²¹¹At is responsible for the parietal cells to secrete gastric acid into the gastric juice [2, 5]. Considering that this secretion also includes ²¹¹At, and is cleared from the stomach wall into the stomach contents, the direct radiation effect in the stomach wall is not considered to be significant, considering the short range of alpha ray in the stomach contents. Alternatively, the testes were considered to be radiosensitive based on the observed histological abnormalities of the high-dose group (50 MBq/kg), as the absorbed dose was reported to be relatively low (83.3 mGy/MBq) in our previous study [5].

As described above, the approximate lethal dose of [²¹¹At]NaAt exceeds 50 MBq/kg under the conditions of this study. No observable adverse effect level (NOAEL) was considered to be less than 5 MBq/kg as the histopathological examination showed the ablation effect of [²¹¹At]NaAt in the thyroid gland of the 5 MBq/kg group. However, no abnormal findings related to the administration of [²¹¹At]NaAt, with the exception of the thyroid, was observed in the 5 MBq/kg group. As inclusion criteria for the FIH clinical trial will include patients with differentiated thyroid cancer who have undergone total thyroidectomy, these individuals will not be affected by thyroid toxicity. In selecting the FIH dose, we will use a common approach to set a start dose at 1/10 the severely toxic dose in 10% of the animals (STD 10) as described in the ICH S9 guidelines [12]. Since the STD 10 of [²¹¹At]NaAt is > 50 MBq/kg from the results of this study, it is reasonable to use 5 MBq/kg in mice as a reference to set the FIH dose.

In the extended single-dose toxicity study of [²¹¹At]NaAt conducted under the reliability standard, no abnormal findings were obtained compared with those of previous studies. Treatment with [²¹¹At]NaAt (50 MBq/kg) at high doses resulted in weight loss, transient bone marrow suppression, and pathological changes in the testis, which require consideration in the FIH clinical trial.